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目的构建靶向乙型肝炎病毒X基因(HBX)的shRNA表达载体pSilencer3.1-shHBX,观察其体外抑制HBX在HepG2细胞中表达的作用,为应用RNA干扰技术进一步研究HBX基因的功能奠定基础。方法设计并构建靶向HBX的shRNA表达载体pSilencer3.1-shHBX,脂质体转染法将HBX表达载体pcDNA3.1-HBX与pSilencer3.1-shHBX共转染人肝癌HepG2细胞,培养72 h后以RT-PCR检测HBX基因表达情况,以Western blot检测HBX蛋白的表达量。结果经酶切和测序鉴定,构建的重组质粒pSilencer3.1-shHBX与设计一致。该质粒使HBX基因mRNA表达量降低47.1%,使HBx蛋白表达量降低58.9%,而阴性对照质粒无此作用。结论成功构建靶向HBX shRNA表达载体pSilencer3.1-shHBX,该质粒可体外抑制HBX在HepG2细胞中的表达。
Objective To construct shRNA expression vector pSilencer3.1-shHBX targeted to hepatitis B virus (HBX) gene and to observe its effect on inhibiting the expression of HBX in HepG2 cells in vitro, so as to lay the foundation for the further study on the function of HBX gene by RNAi technology. Methods shRNA expression vector pSilencer3.1-shHBX targeting HBX was designed and constructed. The human hepatocellular carcinoma HepG2 cells were cotransfected with HBX expression vector pcDNA3.1-HBX and pSilencer3.1-shHBX by lipofectamine 2000. After cultured for 72 h The expression of HBX gene was detected by RT-PCR and the expression of HBX protein was detected by Western blot. Results After digestion and sequencing, the constructed recombinant plasmid pSilencer3.1-shHBX was consistent with the design. The plasmid reduced HBX mRNA expression by 47.1%, decreased HBx protein expression by 58.9%, but negative control plasmid did not. Conclusion The HBX shRNA expression vector pSilencer3.1-shHBX was successfully constructed, which can inhibit the expression of HBX in HepG2 cells in vitro.